A multicellular organism maintains its homeostasis ingeniously by controlling the proliferation and death of its cells. In the course of ontogenesis, many cells are eliminated through apotosis, and even in a mature organism the cells constituting its tissues maintain their functional integrity balancing proliferation against death. Cell death in this context is generally termed "programmed cell death", which is known to occur through the process of apotosis which is morphologically distinguished from that of necrosis, the accidental cell death caused by physical and chemical factors. Apoptotic cell death is characterized by blebbing of the cell membrane, chromatin condensation and DNA fragmentation, with the affected cells being eventually removed by phagocytic cells such as macrophages for reutilization (International Review of Cytology, 68, 251-306, 1980).
Many physiological and pathological events related to apotosis have been identified until now and many attempts made to diagnose, prevent, or treat various diseases through induction or inhibition of the process of apotosis (Science, 267, 1456-1462, 1995). Apotosis is one of the vital phenomena which are attracting more than usual attention in the pharmaceutical industries.
While apotosis is induced in a variety of physiological stimuli, Fas antigen (CD95, APO-1), in particular, is gathering a considerable attention of scientists as a molecule triggering death of immune cells (Science, 267, 1449-1456, 1995). Fas antigen is a 45 kDa type I membrane protein belonging to the TNF (tumor necrosis factor) receptor family and induces cell death by coupling itself to Fas ligand. Whereas Fas antigen is expressed in various blood cells and in many tissues and cells of the liver, heart, and small intestine, expression of Fas ligand, which is a type II membrane protein with a molecular mass of 40 kDa, is confined to activated T lymphocytes, natural killer (NK) cells, macrophages, and certain tissues such as the testis and cornea. Recently, genetic analyses in mice have revealed that the Fas antigen gene has found to be the 1 pr (lymphoproliferation) structural gene which is mutated in mice with autoimmune disease, i.e. 1 pr mice, and that, in gld (generalized-lymphoproliferative disease) mice presenting with symptoms similar to those of 1 pr mice, a mutations of Fas ligand is present. In humans, too, mutations of the Fas antigen gene have been reported in some patients with autoimmune disease, suggesting strongly that disfunction of the Fas/Fas ligand system induces autoimmune diseases (Science, 268, 1347-1349, 1995).
Moreover, it has been found that the human Fas ligand could be cleaved by matrix metalloproteinase and released as the soluble Fas ligand, suggesting that the Fas ligand not only regulates cell-to-cell interactions due to mutual contact but more broadly modulates immune responses (Journal of Experimental Medicine, 182, 1777-1783, 1995).
Among proteins of the TNF family which have various biological activities, TNF-.alpha., lymphotoxin-.alpha.(LT-.alpha.) and lymphotoxin-.beta. (LT-.beta.), have apotosis-inducing activity as well as Fas ligand (The New England Journal of Medicine, 334, 1717-1725, 1996). TNF-.alpha. was first discovered in 1975 as a factor having tumor necrosis-inducing activity but today more importance is attached to TNF-.alpha. as an immunopotentiating factor mediating inflammatory reactions and as a defensive factor against various invasions such as viral and bacterial infections. LT-.alpha. was initially reported as a cytotoxic factor produced by lymphocytes. Like TNF-.alpha., LT-.alpha. forms a homotrimer, binds to the two TNF receptors of 55 kDa and 75 kDa, and although differing in the potency of activity, shows a biological profile similar to that of TNF except for the stimulatory activity of B cell proliferation. LT-.beta. is a 33 kDa membrane-binding protein having high homology with LT-.alpha.; it forms a heterotrimer with LT-.alpha., binds the newly identified LT-.beta. receptor which is different from said two TNF receptors, and has a biological function, i.e. formation of lymph nodes, which is not found in said TNF and LT-.alpha.. More recently, a protein molecule (Apo-2L/TRAIL) having an apotosis-inducing activity and showing high homology with the Fas ligand has been identified for the first time and shown to bind another receptor, termed DR4, different from TNF receptors and Fas antigen to exhibit cytotoxicity (The Journal of Biological Chemistry, 271, 12687-12690, 1996; Science, 276, 111-113, 1997). Therefore, it is supposed that such TNF and TNF receptor family proteins varying in the ligand and receptor expression specificity may have a physiologically different apotosis-inducing function each other.
Recently, it has been reported the relationship of apotosis with diseases (Japanese Journal of Clinical Medicine, 54, No. 7, Jul. 1, 1996). According to this literature, the disease caused by an insufficient apotosis process includes cancer (e.g. mammary cancer, prostatic cancer, ovarian cancer, follicular lymphocytoma, cancer associated with p53 mutation), viral infection (e.g. herpes virus infection, adenovirus infection, poxvirus infection), autoimmune disease (e.g. systemic lupus erythematosus, immune complex glomerulonephritis), etc. On the other hand, the disease caused by an increased apotosis process includes AIDS (acquired immunodeficiency syndrome), neurodegenerative disease (e.g. Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentary retinitis, cerebellar degeneration), diseases due to osteomyelodysplasia (e.g. aplastic anemia), ischemic disease (e.g. myocardial infarction, stroke), toxic liver disease (e.g. alcoholism), etc.
The present invention has for its object to provide a novel Fas ligand-like protein or a partial peptide thereof, or a salt thereof; a DNA coding for the protein; a recombinant gene vector; a transformant which is transformed with said vector; a method of producing the protein; a medicinal composition comprising the protein or DNA; an antibody to the protein, a method of screening for a receptor agonist/antagonist and a kit for the screening; a receptor agonist/antagonist as obtained by the screening and a pharmaceutical composition thereof; a method of screening a compound which promotes or inhibits proteinase having an activity to convert the protein and a kit for the screening; a compound as obtained by the screening and a pharmaceutical composition thereof; a method of screening for a compound which enhances or inhibits a intracellular signal transduction and a kit for the screening; a compound as obtained by the screening and a pharmaceutical composition thereof, and so on.
Isolation of a novel Fas ligand-like protein would cast light on a hitherto-hidden pathway of TNF family/TNF receptor family-mediated apotosis and, should it be expressed with organ- or cell-specificity, would permit a more sophisticated exploration of the relationship of diseases, by organ, with apotosis and lead to development of a novel drug having either an antagonistic action or an agonistic action on the new Fas ligand-like protein and consequently contributing to the prevention and treatment of diseases.
The inventors of the present invention succeeded, after much research, in cloning a cDNA having a novel nucleotide sequence from each of the human liver-, murine embryo- and rat liver-derived cDNA libraries. The inventors further found that the protein encoded by the cloned cDNAs are useful Fas ligand-like proteins. The present invention has been completed on the basis of the above finding and subsequent research.